Targeting the neuromuscular junction for treatment

ABSTRACT

Compositions and methods for targeting therapeutic agents to neuromuscular junctions are disclosed. Also disclosed are methods for treating diseases and conditions affecting the neuromuscular junction. Compositions include a neuromuscular junction targeting peptide coupled to a therapeutic agent. Compositions may further include a linker peptide. Methods for targeting therapeutic agents to neuromuscular junctions and treating diseases and conditions affecting the neuromuscular junction include administering a composition including a neuromuscular junction targeting peptide coupled to a therapeutic agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to provisional patent application No.61/498,707, filed on Jun. 20, 2011, which is incorporated herein byreference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Award No.EY14837 awarded by the National Eye Institute of the National Institutesof Health. The government may have certain rights in the invention.

INCORPORATION OF SEQUENCE LISTING

A paper copy of the Sequence Listing and a computer readable form of thesequence containing the file named “31065-31_ST25.txt”, which is 32,205bytes in size (as measured in MS-DOS), are provided herein and areherein incorporated by reference. This Sequence Listing consists of SEQID NOs: 1-35.

BACKGROUND OF THE DISCLOSURE

The present disclosure relates generally to compositions and methods fortargeting therapeutic agents. More particularly, the present disclosurerelates to compositions and methods useful for targeting therapeuticagents to the neuromuscular junction using neuromuscular junctiontargeting peptides.

The neuromuscular junction is the point at which nerve signals themuscle to contract. More particularly, the neuromuscular junction is thesynapse or junction of the axon terminal of a neuron with a muscle fiberplasma membrane.

Several diseases involve the neuromuscular junction as the primary siteof injury. For example, myasthenia gravis is an autoimmune disorder thatis caused by autoantibodies directed primarily toward the skeletalmuscle acetylcholine receptor (AChR) at the neuromuscular junction. Theantibodies bind to the post-synaptic surface of the neuromuscularjunction and produce a reduction in AChR number and damage the muscleendplate, which leads to a failure of neuromuscular transmission thatresults in muscle weakness. Another category of gravis is caused byantibodies against muscle specific kinase (“MuSK”) at the neuromuscularjunction. Lambert-Eaton syndrome is yet another disorder characterizedby the attack of voltage-gated calcium channels at the neuromuscularjunction by antibodies. Miller Fischer syndrome is another disorderinvolving the attack of nerve terminals by antibodies.

The complement system may underlie one effector mechanism forantibody-mediated immunity, which begins with antibody binding to a cellsurface antigen and the formation of a membrane attack complex. Themembrane attack complex is a multimeric protein complex that producescell lysis and, in the case of myasthenia gravis, destruction of theneuromuscular junction. In antibody-initiated activation of thecomplement cascade, nascent C4b and C3b fragments condense with freehydroxyl and amino groups on biological membranes. Once bound, thesefragments serve as sites for assembly of C4b2a and C3bBb, the centralamplification enzymes of the cascade. Control of their activities toprotect host tissues from autologous complement-mediated injury isthrough a system of cell-associated and serum regulatory proteins.

Complement inhibitors are a class of drugs that show promise fortreating neuromuscular diseases. Complement inhibitors may stop thebody's immune response system from attacking itself. Eculizumab, forexample, is an anti-C5 antibody that is approved for use in paroxysmalnocturnal hemoglobinuria and in Phase 2 trials as a treatment formyasthenia gravis. Eculizumab functions by inhibiting complement.Because administration occurs by infusion, the agent may inhibitcomplement throughout the body.

Another complement inhibitor is rEV576 (OmCI or Conversin). rEV576 is an18.5 kDa recombinantly produced protein derived from tick (Ornithodorosmoubata) saliva that specifically inhibits C5 complement. rEV576 appearsto directly bind C5 to prevent interaction with C5 convertase.Administration of rEV576 has been shown to reduce serum complementactivity, diminish C9 deposition at the neuromuscular junction, andreduce cytotoxicity of serum from treated animals.

Therapies for myasthenia gravis generally focus on enhancingneuromuscular transmission by inhibition of cholinesterase using agentssuch as pyridostigmine. Other treatments such as corticosteroids,azathioprine, tacrolimus, and mycophenolate, are directed to suppressingor modulating the immune system. Acute exacerbations of weakness may betreated by plasmapheresis or intravenous immunoglobulins. Whileeffective, these treatments can be expensive and may entail side affectsthat affect organ systems beyond the neuromuscular junction. Thesetreatments may additionally result in systemic side effects becauseadministration occurs throughout the body. The immunotherapies are notspecifically focused on myasthenia gravis, but rather generally moderateimmune response through reduction of autoantibody levels directly orindirectly through suppression of B and T cell activity.

Although treatments are available for conditions resulting fromneuromuscular junction injury, there remains a concern over theirefficacy, side-effects, and/or costs. Moreover, complement inhibitorstrategies rely on systemic inhibition of complement. Accordingly, thereexists a continued need to develop alternative treatments and methodsfor treating conditions resulting from neuromuscular junction injury.

SUMMARY OF THE DISCLOSURE

The present disclosure relates generally to compositions and methods fortargeting therapeutic agents. More particularly, the present disclosurerelates to compositions and methods useful for targeting therapeuticagents to the neuromuscular junctions using neuromuscular junctiontargeting peptides.

In one aspect, the present disclosure is directed to compositionsincluding a neuromuscular junction targeting peptide coupled to atherapeutic agent.

In another aspect, the present disclosure is directed to a method ofdelivering a therapeutic agent to a neuromuscular junction. The methodincludes administering a composition that includes a neuromuscularjunction targeting peptide coupled to a therapeutic agent.

In another aspect, the present disclosure is directed to methods fortreating a neuromuscular junction-related disease or condition. Themethod includes administering to a subject in need thereof a compositionthat includes a neuromuscular junction targeting peptide coupled to atherapeutic agent.

In another aspect, the present disclosure is directed to a recombinantnucleic acid construct encoding a neuromuscular junction targetingpeptide operably linked to a heterologous nucleic acid sequence encodinga therapeutic polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosure will be better understood, and features, aspects andadvantages other than those set forth above will become apparent whenconsideration is given to the following detailed description thereof.Such detailed description makes reference to the following drawings,wherein:

FIG. 1 is a schematic showing the domain structure of the laminin-rEVpolypeptide as described in Example 1.

FIG. 2 is a map of the pET28a expression vector.

FIG. 3 is an SDS-gel showing the expression of the laminin-rEVpolypeptide in BL21 as described in Example 1.

FIG. 4 is a Western blot of purified rEV, expressed rEV from inducedcells, expressed laminin-rEV (LrEV) from induced cells, and uninducedcells.

FIG. 5 is a schematic showing the domain structure of the HIV-rEVpolypeptide as described in Example 2.

FIG. 6 is a schematic showing the domain structure of the RVG-rEVpolypeptide as described in Example 3.

FIG. 7 is a schematic showing the construction of the pET16b-scFv-DAFexpression vector as described in Example 4.

FIG. 8 is a photomicrograph showing GFP expression in BHK-21 cellstransfected with the IgGsp-V_(H)-V_(L) as described in Example 4.

FIG. 9 is an SDS-gel showing the expression and purification of scFv-DAFas described in Example 4.

FIG. 10 is a graph showing the specificity binding of scFv-DAF andscFv1956 to hAChRα1l-210 polypeptides as described in Example 5.

FIG. 11 is a graph showing complement-mediated haemolysis of sheeperythrocytes incubated with scFv-DAF as described in Example 6.

FIG. 12 are photomicrographs showing localization of the scFv-35-DAF tothe neuromuscular junction in diaphragms of mice as described in Example7.

FIG. 13 shows immunofluorescence micrographs showing scFv-DAF reductionof C3 deposits on TE671 cells as described in Example 8.

FIG. 14 is a schematic showing the domain structures of variousneuromuscular junction targeting constructs according to the presentdisclosure.

FIG. 15 is a graph depicting the therapeutic effect of scFv and scFv-DAFin EAMG mice as described in Example 9.

FIG. 16 is a graph is a graph depicting the therapeutic effect of scFvand scFv-DAF in EAMG Lewis rats as described in Example 9.

While the disclosure is susceptible to various modifications andalternative forms, specific embodiments thereof have been shown by wayof example in the drawings and are herein described below in detail. Itshould be understood, however, that the description of specificembodiments is not intended to limit the disclosure to cover allmodifications, equivalents and alternatives falling within the spiritand scope of the disclosure as defined by the appended claims.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the disclosure belongs. Although any methods andmaterials similar to or equivalent to those described herein can be usedin the practice or testing of the present disclosure, the preferredmethods and materials are described below.

In accordance with the present disclosure, compositions and methods havebeen discovered that allow for targeting neuromuscular junctions. Thecompositions and methods have significant impact as they allow for thetargeted delivery of therapeutic agents to the neuromuscular junction.The compositions and methods further allow for the treatment ofneuromuscular junction-related diseases and conditions such as, forexample, myasthenia gravis, experimentally acquired myasthenia gravis,Lambert-Eaton syndrome, and Miller Fischer syndrome in whichneuromuscular junctions may be affected.

Compositions

In one aspect, the present disclosure is directed to a compositionincluding a neuromuscular junction targeting peptide (“NMJTP”) coupledto a therapeutic agent (“TA”) (see e.g., FIG. 14). The terms“polypeptide,” “peptide” and “protein” are used interchangeably hereinto refer to a polymer of amino acid residues of any length, unlessindicated otherwise.

As used herein, “coupled to” refers to a composition wherein theneuromuscular junction targeting peptide is directly or indirectlyattached to, fused with, joined to, and/or linked to the therapeuticagent. For example, if the composition is prepared using knownrecombinant protein expression methods, a nucleic acid sequence encodingthe neuromuscular junction targeting peptide may be joined to a nucleicacid sequence encoding the therapeutic agent. In such an example, theneuromuscular junction targeting peptide would be directly coupled tothe therapeutic agent. In another example, the nucleic acid sequenceencoding the neuromuscular junction targeting peptide may be indirectlyjoined to a nucleic acid sequence encoding the therapeutic agent byincluding at least one linker between the neuromuscular junctiontargeting peptide and the therapeutic agent.

The compositions of the present disclosure may be prepared as part of alarger construct that is subjected to further processing to produce thefinal composition having the neuromuscular junction targeting peptidecoupled to the therapeutic agent. Domain structures of larger constructsare illustrated in FIG. 14. In one embodiment, for example, theconstruct may include an ATG start site coupled to an affinity tagcoupled to a protease cleavage site (“Protease Site”) coupled to aneuromuscular junction targeting peptide (“NMJTP”) coupled to a linkercoupled to a therapeutic agent (“TA”) (see, FIG. 14). In anotherembodiment, the domain structure may include an ATG start site coupledto an affinity tag coupled to a protease cleavage site coupled to aNMJTP coupled to a linker coupled to a NMJTP coupled to a linker coupledto a TA (see, FIG. 14). Table 1 summarizes the neuromuscular junctiontargeting peptide indicated in FIG. 14 and their specificities (bindingtarget).

TABLE 1 Neuromuscular Junction Targeting Peptides. NMJTP SpecificityMcAb35 Muscle nicotinic acetylcholine receptor AE-2 AcetylcholinesteraseAE-3 Acetylcholinesterase C1B7 Acetylcholinesterase HB-189 Neuronalacetylcholine receptor HB8987 Neuronal acetylcholine receptor alphasubunit

The therapeutic agent may be coupled to either the N-terminus orC-terminus of the neuromuscular junction targeting peptide. Afterfurther processing as further described herein, for example, thecomposition may have the structure NMJTP-TA, in which the therapeuticagent is coupled to the C-terminus of the neuromuscular junctiontargeting peptide. In another example, the composition may have thestructure TA-NMJTP after further processing, in which the therapeuticagent is coupled to the N-terminus of the neuromuscular junctiontargeting peptide. Similarly, the neuromuscular junction targetingpeptide may be coupled to a linker at either the N-terminus, theC-terminus, or both the N-terminus and C-terminus of the neuromuscularjunction targeting peptide (see, FIG. 14). When a linker is included inthe composition, the linker is positioned between the neuromuscularjunction targeting peptide and the therapeutic agent. In one embodiment,the composition may have the structure: NMJTP-linker-TA after furtherprocessing. In another embodiment, the composition may have thestructure: TA-linker-NMJTP after further processing. In yet anotherembodiment, the composition may have the structure:NMJTP-linker-NMJTP-linker-TA after further processing. In still anotherembodiment, the composition may have the structure:TA-linker-NMJTP-linker-NMJTP after further processing.

As described herein, the composition may be subjected to furtherprocessing. For example, the presence of an affinity tag allows forpurification via interaction between the affinity tag and an affinitysubstrate as known by those skilled in the art and described herein. Thecomposition may also be subjected to treatment with a protease thatcleaves the composition at a position between the affinity tag and theneuromuscular junction targeting peptide. Protease treatment allows forthe preparation of a composition including a neuromuscular junctiontargeting peptide and a therapeutic agent, which may further include atleast one linker.

Neuromuscular Junction Targeting Peptides

The composition of the present disclosure includes a neuromuscularjunction targeting peptide (“NMJTP”). The neuromuscular junctiontargeting peptide may be a peptide molecule that binds to or interactswith a molecule located at or near the neuromuscular junction. Forexample, the molecule may be located at the presynaptic or postsynapticside of the neuromuscular junction. Suitable neuromuscular junctiontargeting peptides may bind to or interact with a molecule present on aneuron at or near the neuromuscular junction. Other suitableneuromuscular junction targeting peptides may bind to or interact with amolecule present on a muscle cell at or near the neuromuscular junction.The neuromuscular junction targeting peptides have a binding affinityfrom about 0.5 nM to about 50 μM. A particularly suitable bindingaffinity may be, for example, from about 1 nM to about 40 μM. A evenmore suitable binding affinity may be, for example, from about 0.1 μM toabout 0.75 μM. Binding of a neuromuscular targeting peptide may bedetermined by methods known by those skilled in the art. Suitablemethods for determining binding affinity may be, for example,enzyme-linked immunosorbent assay (ELISA), Western blot analysis, andimmunostaining. Neuromuscular junction targeting peptides alone orcoupled to therapeutic agents may also be prepared using, for example,recombinant protein expression methods to further include a detectablelabel such as, for example, a fluorescent tag, such that binding may bedirectly detected. Analysis of immunostained or directly labeledcompositions may be further analyzed by fluorescent pixel analysis, forexample, to determine binding affinity.

Suitable neuromuscular junction targeting peptides may be, for example,peptides obtained from larger protein molecules. Other suitableneuromuscular junction targeting peptides may be, for example, antibodymolecules. Particularly suitable antibody molecules may be, for example,antibody variable regions, antibody heavy chains, antibody light chains,Fab, F(ab′)₂, F(ab′) and single chain antibodies (scFv).

Suitable neuromuscular junction targeting peptides may be obtained fromproteins such as, for example, laminin, a zinc finger domain of thehuman immunodeficiency virus (HIV) nucleocapsid protein, rabies virusglycoprotein (RVG), α-bungarotoxin, agrin, antibodies to theacetylcholine receptor, antibodies to acetylcholinesterase, musclespecific kinase, calcium channels, and sodium channels. Particularlysuitable neuromuscular junction targeting peptides may be, for example,a laminin peptide, a human immunodeficiency virus peptide such as, forexample, a zinc finger domain of the HIV nucleocapsid protein, a rabiesvirus glycoprotein peptide, an α-bungarotoxin peptide, an agrin peptide,a single chain antibody peptide that specifically binds toacetylcholinesterase and a single chain antibody peptide thatspecifically binds to acetylcholine receptor. Suitable neuromuscularjunction targeting peptides may also be non-peptide chemicals, such asnicotine. Particularly suitable neuromuscular junction targetingpeptides may be, for example, those having an amino acid sequence of SEQID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, andSEQ ID NO: 9.

Therapeutic Agents

The composition of the present disclosure includes a therapeutic agentthat is coupled to the neuromuscular junction targeting peptide. As usedherein, the term “therapeutic agent” refers to any type of drug,medicine, pharmaceutical, hormone, antibiotic, protein, gene, growthfactor, bioactive material, etc., used for treating, controlling, orpreventing diseases or medical conditions. As used herein, the term“derivative” refers to a compound or peptide obtained from anothercompound (e.g., a lead or parent compound). The therapeutic agent may bea protein that may be recombinantly expressed such that a nucleic acidsequence encoding an amino acid sequence corresponding to a therapeuticpolypeptide may be operably linked to a nucleic acid sequence encodingan amino acid sequence corresponding to a neuromuscular junctiontargeting peptide and/or a nucleic acid sequence encoding an amino acidsequence corresponding to a linker.

Suitable therapeutic agents may be, for example, complement inhibitors,acetylcholinesterase inhibitors, trophic agents, and paralytic agents.Suitable complement inhibitors may be, for example, decay acceleratingfactor (“DAF”; “CD55”)), rEV576, complement receptor 1 (“CR1”; “CD35”),membrane cofactor protein (“MCP”; “CD46”), compstatin, compstatinderivatives, POT-4, C1 inhibitor, C4b-binding protein (“C4BP”), factor H(“FH”), complement receptor Ig (“CRIg”), CD59, clusterin, C3-inhibitors,peptide 2J, human beta-defensin 2, CRIT-H17, Ac-SHLGLAR-H, Ac-RLLLAR-H,C1s-INH-248, S-protein, and Crry. Other suitable therapeutic agents maybe, for example, peptides obtained from full length complementinhibitors. For example, control protein repeats of DAF may be used asthe therapeutic agent. Other suitable therapeutic agents such aschemicals and peptidomimetics, that are not peptides, but inhibitcomplement may be, for example, circumin, W-54011, NGD 2000-1,NDT9520492, CP-447,697, NDT 9513727, SB290157, SB290157(A), SB290157(B),BCX1470, a C1s inhibitor, BCX1470, PMX53, PMX205, C089, JPE1375.

Particularly suitable therapeutic agents may be, for example, thosehaving an amino acid sequence of SEQ ID NO: 11 and SEQ ID NO: 12.

Linkers

Additionally or alternatively, the composition may include at least onelinker such that the neuromuscular junction targeting peptide isindirectly coupled to the therapeutic agent (see, FIG. 14). In oneembodiment, the composition may include a linker positioned between theneuromuscular junction targeting peptide (“NMJTP”) and the therapeuticagent (“TA”) (see, FIG. 14). In another embodiment, the composition mayinclude a linker positioned between two domains of the neuromuscularjunction targeting peptide such as, for example, when the compositionuses a single chain antibody wherein the first linker is positionedbetween a V_(H) and a V_(L) of the single chain antibody (see, FIG. 14).In embodiments using a single chain antibody as the neuromuscularjunction targeting peptide, the two variable regions of the antibody areseparated by a linker to allow proper folding so binding to the targetedprotein may occur. For example, if the composition is prepared usingknown recombinant protein expression methods, a nucleic acid sequenceencoding the neuromuscular junction targeting peptide may be coupled toa nucleic acid sequence encoding the linker that may be coupled to anucleic acid sequence encoding the therapeutic agent.

Without intending to be bound by theory, it is believed that a linkerprovides additional distance or separation between the neuromusculartargeting peptide and the therapeutic agent, or also, for example, tolimit possible steric hindrance that may otherwise interfere with theactivity of the therapeutic agent or targeting by the neuromuscularjunction targeting peptide. Linkers may further allow for may furtherallow for proper folding of the neuromuscular junction targetingpeptides and/or therapeutic agents. Nucleotide sequences encodingparticularly suitable linkers may be, for example, those shown in Table2.

TABLE 2 Linker Sequences. SEQ ID Nucleotide NO: Description LengthSequence — 2 aa GS linker 6 ggcagc 19 6 aa [GS]x 18 ggtagcggcagcggtagclinker 20 10 aa [GS]x 30 ggtagcggcagcggtagcggtagcggcagc linker 2110 aa flexible 30 ggtgaaaatttgtattttcaatctggtggt protein domain linker22 8 aa protein 24 tccgcttgttactgtgagctttcc domain linker 23 Split 51cgaccagcctgtaagattccaaatgacctgaagcagaaagttatgaatc fluorophore aclinker; Freiburg standard 24 15 aa flexible 45ggtggaggaggttctggaggcggtggaagtggtggcggaggtagc GS linker; Freiburgstandard 25 Short Linker 12 ggtggttctggt (Gly-Gly-Ser- Gly) 26Middle Linker 24 ggtggttctggtggtggttctggt (Gly-Gly-Ser- Gly)x2 27Long Linker 36 ggtggttctggtggtggttctggtggtggttctggt (Gly-Gly-Ser- Gly)x328 GSAT Linker 108 ggtggttctgccggtggctccggttctggctccagcggtggcagctctggtgcgtccggcacgggtactgcgggtggcactggcagcggttccggt actggctctggc 29SEG-Linker 108 ggtggttctggcggcggttctgaaggtggcggctccgaaggcggcggcagcgagggcggtggtagcgaaggtggtggctccgagggtggcggt tccggcggcggtagc 30Z-EGFR- 192 gtggataacaaatttaacaaagaaatgtgggcggcgtgggaagaaatt 1907_Short-cgtaacctgccgaacctgaacggctggcagatgaccgcgtttattgcga Linkergcctggtggatgatccgagccagagcgcgaacctgctggcggaagcgaaaaaactgaacgatgcgcaggcgccgaaaaccggcggtggttctg gt 31 Z-EGFR-gtggataacaaatttaacaaagaaatgtgggcggcgtgggaagaaatt 1907_Middle- 204cgtaacctgccgaacctgaacggctggcagatgaccgcgtttattgcga Linkergcctggtggatgatccgagccagagcgcgaacctgctggcggaagcgaaaaaactgaacgatgcgcaggcgccgaaaaccggcggtggttctg gtggtggttctggt 32Z-EGFR- 216 gtggataacaaatttaacaaagaaatgtgggcggcgtgggaagaaattc 1907_Long-gtaacctgccgaacctgaacggctggcagatgaccgcgtttattgcgag Linkercctggtggatgatccgagccagagcgcgaacctgctggcggaagcgaaaaaactgaacgatgcgcaggcgccgaaaaccggcggtggttctggt ggtggttctggtggtggttctggt33 (Gly4Ser)3 45 ggtggaggaggctctggtggaggcggtagcggaggcggagggtcg FlexiblePeptide Linker

Pharmaceutical Formulations

The composition of the present disclosure may also includepharmaceutical formulations. Pharmaceutical formulations may include,for example, pharmaceutically acceptable salts, carriers, adjuvants,vehicles, oils, and lipids as known by those skilled in the art. Thepharmaceutical formulations may also be, for example, tablets, capsules,ingestible liquids, powders, liposomes, nanoparticles and controlledrelease formulations as known by those skilled in the art.

Recombinant Proteins, Vectors, Host Cells and Expression

The compositions of the present disclosure may be prepared asrecombinant proteins using recombinant protein expression methods.Suitable polynucleotides may be, for example, SEQ ID NO: 13(laminin-rEV), SEQ ID NO: 15 (HIV-rEV), and SEQ ID NO: 17 (RVG-rEV).These too permit a degree of variability in their sequence, as forexample due to degeneracy of the genetic code, codon bias in favor ofthe host cell expressing the polypeptide, and conservative amino acidsubstitutions in the resulting protein. Consequently, the polypeptidesand constructs of the invention include not only those which areidentical in sequence to the above sequence but also those variantpolypeptides with the structural and functional characteristics thatremain substantially the same.

Such variants (or “analogs”) may have a sequence homology (“identity”)of 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more with the referencesequence. In this sense, techniques for determining amino acid sequence“similarity” are well known in the art. In general, “similarity” meansthe exact amino acid to amino acid comparison of two or morepolypeptides at the appropriate place, where amino acids are identicalor possess similar chemical and/or physical properties such as charge orhydrophobicity. A so-termed “percent similarity” may then be determinedbetween the compared polypeptide sequences. Techniques for determiningnucleic acid and amino acid sequence identity also are well known in theart and include determining the nucleotide sequence of the mRNA for thatgene (usually via a cDNA intermediate) and determining the amino acidsequence encoded therein, and comparing this to a second amino acidsequence. In general, “identity” refers to an exact nucleotide tonucleotide or amino acid to amino acid correspondence of twopolynucleotides or polypeptide sequences, respectively. Two or morepolynucleotide sequences can be compared by determining their “percentidentity”, as can two or more amino acid sequences. Programs availablesuch as, for example, BLAST, are capable of calculating both theidentity between two polynucleotides and the identity and similaritybetween two polypeptide sequences, respectively. Other programs forcalculating identity or similarity between sequences are known in theart.

The present disclosure is further directed to vectors including nucleicacid sequences encoding the compositions including a neuromuscularjunction targeting peptide. The term “vector”, as used herein, refers toany recombinant polynucleotide construct that may be used to introduceheterologous DNA into a host cell. Vectors of the present disclosure mayfurther include nucleic acid sequences encoding a neuromuscular junctiontargeting peptide operably linked to a nucleic acid sequence encoding atherapeutic agent. Vectors of the present disclosure may further includenucleic acid sequences encoding a neuromuscular junction targetingpeptide operably linked to a nucleic acid sequence encoding a linkerthat is further operably linked to a nucleic acid sequence encoding atherapeutic agent.

The compositions of the present disclosure may be produced inprokaryotic and eukaryotic cells using expression vectors suitable forthe particular host cell. Particularly suitable prokaryotic cells maybe, for example, Escherichia coli and Salmonella sp. Particularlysuitable eukaryotic cells may be, for example, mammalian cells, insectcells, and yeast cells.

The term “construct”, as used herein, refers to any recombinantpolynucleotide molecule. Examples of constructs may be a plasmid, acosmid, a virus, an autonomously replicating polynucleotide molecule, aphage, or a linear or circular single-stranded or double-stranded DNA orRNA polynucleotide molecule, derived from any source, capable of genomicintegration or autonomous replication, comprising a polynucleotidemolecule where one or more polynucleotide molecule(s) has been linked ina functionally operative manner, i.e., operably linked.

As used herein, “operably linked” refers to the joining of nucleic acidsequences such that one sequence can provide a required function to alinked sequence. In the context of a promoter, “operably linked” meansthat the promoter is connected to a sequence of interest such that thetranscription of that sequence of interest is controlled and regulatedby that promoter. When the sequence of interest encodes a protein andwhen expression of that protein is desired, “operably linked” means thatthe promoter is linked to the sequence in such a way that the resultingtranscript will be efficiently translated. If the linkage of thepromoter to the coding sequence is a transcriptional fusion andexpression of the encoded protein is desired, the linkage is made sothat the first translational initiation codon in the resultingtranscript is the initiation codon of the coding sequence.Alternatively, if the linkage of the promoter to the coding sequence isa translational fusion and expression of the encoded protein is desired,the linkage is made so that the first translational initiation codoncontained in the 5′ untranslated sequence associated with the promoterand is linked such that the resulting translation product is in framewith the translational open reading frame that encodes the desiredprotein. Nucleic acid sequences that can be operably linked may be, forexample, sequences that provide gene expression functions (i.e., geneexpression elements such as promoters, 5′ untranslated regions, introns,protein coding regions, 3′ untranslated regions, polyadenylation sites,and/or transcriptional terminators), sequences that provide DNA transferand/or integration functions, sequences that provide for selectivefunctions (i.e., antibiotic resistance markers, biosynthetic genes),sequences that provide scoreable marker functions (i.e., reportergenes), sequences that facilitate in vitro or in vivo manipulations ofthe sequences (i.e., polylinker sequences, site specific recombinationsequences) and sequences that provide replication functions (i.e.,bacterial origins of replication, autonomous replication sequences,centromeric sequences). Additional sequences that may be operably linkedmay be, for example, sequences that facilitate purification of therecombinantly expressed protein such as, for example, affinity tags.Other additional sequences that may be operably linked may be, forexample, sequences that encode protease cleavage sites.

An “isolated” polynucleotide (e.g., an “isolated DNA” or an “isolatedRNA”) refers to a polynucleotide at least partially separated from atleast some of the other components of the naturally occurring organismsuch as, for example, the cell structural components or otherpolypeptides or nucleic acids commonly found associated with thepolynucleotide.

An “isolated” polypeptide refers to a polypeptide that is at leastpartially separated from at least some of the other components of thenaturally occurring organism such as, for example, the cell orstructural components or other polypeptides or nucleic acids commonlyfound associated with the polypeptide.

Once the vector has been constructed by operably linking the components,it may be introduced into a host cell. Operably linking thepolynucleotide sequence encoding a neuromuscular junction targetingpeptide to a polynucleotide sequence encoding a therapeutic agent and/ora linker, as provided by the present disclosure, results in theproduction of an expressed polypeptide from the host cell. The vectormay be introduced into the host cell by methods known by those skilledin the art. Suitable methods may be, for example, transfection,transformation, and electroporation. The host cell is then culturedunder suitable conditions to permit expression of the composition, whichis then recovered by isolation and/or purification.

Recombinantly expressed compositions according to the present disclosuremay be further isolated and purified according to methods known by thoseskilled in the art. For example, protein purification may be performedby affinity chromatography, precipitation, chromatography (e.g.,affinity, ion exchange, HPLC, gel filtration), extraction,ultrafiltration, electrophoresis, and combinations thereof Proteinisolation and purification may be facilitated by including sequences andamino acid motifs or use of protein expression vectors having sequencesand amino acid motifs for affinity purification. Suitable sequences andmotifs may be, for example, histidine tags, antigen peptide tags, chitinbinding protein, maltose binding protein, glutathione-S-transferase andother suitable tags known by those skilled in the art. Particularlysuitable affinity tags may be, for example, histidine tags (“His tags”)as illustrated in FIG. 14. As known by those skilled in the art,affinity tags aid in the purification of the protein containing theaffinity tag.

Recombinantly expressed compositions according to the present disclosuremay further include cleavage sites for proteases to further isolate thecomposition from other domains. Suitable protease cleavage sites may beany protease cleavage site known by those skilled in the art. Suitableproteases may be, for example, Factor Xa, enterokinase, thrombin, TEVprotease (Invitrogen, Carlsbad, Calif.), PRESCISSION (GE Healthcare LifeSciences, Piscataway, N.J.), pepsin cleavage sites, trypsin cleavagesites, chymotrypsin cleavage sites, thermolysin cleavage sites, andother protease cleavage sites known by those skilled in the art.Particularly suitable protease cleavage sites may be, for example, athrombin cleavage site.

Additionally or alternatively, the compositions of the presentdisclosure may be prepared using chemical synthesis methods. Suitablechemical synthesis methods are well-known in the art and may include,for example, liquid-phase peptide synthesis, solid-phase peptidesynthesis, fragment condensation, and chemical ligation.

Methods for Delivering a Therapeutic Agent to the Neuromuscular Junction

In another aspect, the present disclosure is directed to a method fordelivering a therapeutic agent to the neuromuscular junction. The methodincludes administering a composition including a neuromuscular junctiontargeting peptide coupled to a therapeutic agent.

The neuromuscular junction targeting peptide may be any neuromuscularjunction targeting peptide as described herein. The therapeutic agentmay be any therapeutic agent as described herein. The composition mayfurther include at least one linker as described herein.

Administration may be to a subject. Suitable methods for administrationto a subject may be, for example, by intravenous injection, intravenousinfusion, intraperitoneal injection, intradermal injection,intramuscular injection, subcutaneous injection, intranasal, oral, andother methods known by those skilled in the art. Administration may alsobe to a cell in culture. Suitable methods for administration to a cellin culture may also, for example, by pipetting, pouring a solutioncontaining the composition, and other methods know in the art.

Dosage of the composition to be administered may be determined by thoseskilled in the art. Dosage may depend on various factors such as, forexample, the condition or disease, weight of the subject, age of thesubject, method of administration, route of administration, whetheradministered for an in vivo purpose, whether administered for an invitro purpose, and other factors. The dosage to a subject will generallyinclude an amount that is sufficient to provide some improvement orbenefit to the subject so as to provide some alleviation, mitigation, ordecrease in at least one clinical symptom in the subject. The dosage mayalso include an amount that is sufficient to provide an in vitrocomplement inhibitory effect or prevention of complement-dependent celllysis, for example.

Suitable dosages may be from about 0.001 μg/ml to about 4 μg/ml.Particularly suitable dosages may be from about 0.001 μg/ml to about0.02 μg/ml. Suitable dosages may be determined in vitro, for example, byinvestigating the inhibition of complement hemolytic activity ofantibody sensitized erythrocytes, by luminescent bioassay ofantibody-initiated, complement mediated injury of cell lines (forexample, the toxilight bioassay commercially available from Cambrex,Rockland, Me.). Suitable dosages may also be determined in vivo, forexample, by analyzing targeting to the neuromuscular junction, analyzingthe production of weakness in the subject, and determining alterationsin systemic complement activity. Those skilled in the art willappreciate that the therapeutic effects need not be complete orcurative, as long as some benefit is provided to the subject.

Methods for Treating Neuromuscular Junction-Related Diseases orConditions

In another aspect, the present disclosure is directed to a method fortreating a neuromuscular junction-related disease or condition. Themethod includes administering to a subject in need thereof a compositionincluding a neuromuscular junction targeting peptide coupled to atherapeutic agent.

As used herein “neuromuscular junction-related diseases or conditions”refer to diseases or conditions resulting from injury at and/or to theneuromuscular junction. A neuromuscular junction-related disease orcondition may be, for example, myasthenia gravis, experimentallyacquired myasthenia gravis, Lambert-Eaton syndrome, Miller Fischersyndrome, congenital myasthenic syndromes, botulism, organophosphatepoisoning, and other toxins that compromise the neuromuscular junction.

The methods include the administration of the compositions to a subjectin need thereof including individuals afflicted with neuromuscularjunction-related diseases or conditions resulting from injury at and/orto the neuromuscular junction as described herein. Additionally, asubject in need thereof includes laboratory animals experimentallyinduced to mimic diseases or conditions resulting from injury at and/orto the neuromuscular junction, thus serving as animal models of thesediseases and conditions. As such, in some embodiments of the presentdisclosure, the methods disclosed herein are directed to a subset of thegeneral population such that not all of the general population maybenefit from these methods.

Suitable subjects may be mammals. Suitable mammals may be, for example,humans, mice, rats, rabbits, guinea pigs, and monkeys.

The neuromuscular junction targeting peptide may be any neuromuscularjunction targeting peptide described herein. The therapeutic agent maybe any therapeutic agent as described herein. The composition mayfurther include at least one linker as described herein.

Suitable methods for administration may be, for example, by intravenousinjection, intravenous infusion, intraperitoneal injection, intradermalinjection, intramuscular injection, subcutaneous injection, intranasal,oral, and other methods known by those skilled in the art.

Suitable therapeutically effective amounts may be, for example, fromabout 1 ng/kg to about 0.1 mg/kg. More particularly, the therapeuticallyeffective amount may be about 5 mg/kg. Suitable therapeuticallyeffective amounts may further be described as having a half maximalinhibitory concentration (IC₅₀) of from about 0.001 mg/ml to about 40mg/ml. A particularly suitable IC₅₀ may be from about 10 ng/ml to about20 mg/ml. The therapeutically effective amount may be characterized, forexample, by observing a prolonged biological effect without observationof the production of weakness in the subject. The therapeuticallyeffective amount of the composition to be administered to the subjectmay be determined by those skilled in the art. The therapeuticallyeffective amount may depend on various factors such as, for example, thecondition or disease, weight of the subject, age of the subject, methodof administration, route of administration, and other factors.Generally, the therapeutically effective amount will include an amountthat is sufficient to provide some improvement or benefit to the subjectso as to provide some alleviation, mitigation, or decrease in at leastone clinical symptom in the subject. Those skilled in the art willappreciate that the effect need not be complete or curative, as long assome benefit is provided to the subject.

The disclosure will be more fully understood upon consideration of thefollowing non-limiting Examples.

EXAMPLES Example 1 L-rEV Cloning and Expression

In this Example, a composition including the neuromuscular junctiontargeting peptide obtained from laminin (encoded by SEQ ID NO: 1)coupled to rEV (encoded by SEQ ID NO: 10) was prepared by recombinantprotein expression.

Specifically, the laminin neuromuscular junction targeting peptide wascoupled to rEV. As shown in FIG. 1, the construct further included alinker, a His tag and a thrombin cleavage site. Codon optimization wasperformed using the codon optimization program available from IntegratedDNA Technologies.

The cDNA encoding laminin-linker-rEV was subcloned into the prokaryoticexpression vector plasmid pET-28-a(+) via Nhel and EcoRI sites withinmulti-cloning sites. The plasmid was verified by restriction enzymeanalysis and direct sequencing. A schematic of the plasmid construct isshown in FIG. 2.

For recombinant protein expression of the laminin-linker-rEV, BL21 E.coli were transformed with the pET28a-laminin-linker-rEV plasmidprepared above and induced with 1 mM 1 mMisopropyl-β-D-thiogalactopyranoside (IPTG) for 4 hrs. Cells were lysedby sonication in lysis buffer containing 3.2 mM Na₂HPO₄, 0.5 mM KH₂PO₄,1.3 mM KCl, 135 mM NaCl, pH 7.4, 5 mM imidazole, lysozyme and proteaseinhibitor tablet (Roche). After centrifugation at 13,000 rpm at 4° C.for 20 minutes, the soluble fraction was applied to a Talon metalaffinity column (Clontech), washed with buffer A (3.2 mM Na₂HPO₄, 0.5 mMKH₂PO₄, 1.3 mM KCl, 135 mM NaCl, pH 7.4) and eluted with buffer Acontaining different concentrations of Imidazole (20 mM to 500 mM). Thefractions containing the protein of interest were pooled andconcentrated using Amicon ultra 10K MWCO filters (Millipore). Theconcentrated protein was then applied onto a Superdex200 column (GEhealthcare) for gel filtration chromatography. The eluted fractions wereanalyzed for the presence of protein and concentration of positivefractions was undertaken. The resultant protein was stored at −80° C. orused for further experimentation.

Concentration of the purified protein was determined by the Bradfordassay. As shown in FIG. 3, induced BL21 cells began expressinglaminin-rEV within 1 hour after induction with maximal expression at 4hours. Western blot analysis using an anti-rEV antibody confirmedexpression in induced cells. See, FIG. 4.

Example 2 HIV-rEV

In this Example, a composition including the neuromuscular junctiontargeting peptide from HIV (encoded by SEQ ID NO: 3) coupled to rEV(encoded by SEQ ID NO: 10) was prepared as set forth in Example 1.

Specifically, the HIV neuromuscular junction targeting peptide wascoupled to rEV. As shown in FIG. 5, the construct further included alinker, a His tag and a thrombin cleavage site. Codon optimization wasperformed using the codon optimization program available from IntegratedDNA Technologies.

BL21 E. coli were transformed with the pET28a-HIV-linker-rEV plasmid andinduced with IPTG for 4 hours. Cells were lysed and the protein waspurified as set forth in Example 1 above. The resultant protein wasstored at −80° C. or used for further experimentation.

Example 3 RVG-rEV

In this Example, a composition including the neuromuscular junctiontargeting peptide obtained from RVG (encoded by SEQ ID NO: 5) coupled torEV (encoded by SEQ ID NO: 10) was prepared as set forth in Example 1.

Specifically, the RVG neuromuscular junction targeting peptide wascoupled to rEV. As shown in FIG. 6, the construct further included alinker, a His tag and a thrombin cleavage site. Codon optimization wasperformed using the codon optimization program available from IntegratedDNA Technologies.

BL21 E. coli were transformed with the pET28a-RVG-linker-rEV plasmid andinduced with IPTG for 4 hours. Cells were lysed and the protein waspurified as set forth in Example 1 above. The resultant protein wasstored at −80° C. or used for further experimentation.

Example 4

In this Example, a composition including the neuromuscular junctiontargeting peptide using the single chain antibody fragment (scFv)obtained from Mab 35 coupled to decay accelerating factor (DAF) wasprepared as set forth in Example 1.

Specifically, the scFv was fused to DAF and inserted into the pET16bexpression vector as outlined in FIG. 7. To generate the V_(H)-V_(L)fragment of the scFv, RNA was obtained from the TIB-175 hybridoma cellline (obtained from ATCC), which secretes the Mab 35 antibody. Thevariable heavy chain (V_(H)) and/or variable light chain (V_(L)) werethe amplified by RT-PCR. Overlapping PCR was used to generate theV_(H)-V_(L) fragment with the (GGGGS)₃ (SEQ ID NO: 34) linker to producethe single chain AChR antibody scFv. Sequencing confirmed the identityof the cloned fragments as V_(H) and V_(L) regions. Two signalingpeptide constructs were then produced using the CD59 fragment or the IgGsignal peptide. The CD59sp- or IgGsp-signaling peptide was then fused tothe V_(H)-V_(L) fragment and cloned into the pIRES2-AcGFP1 vector(Clonetech, Mountain View, Calif.). BHK-21 cells were transfected withthese constructs. FIG. 8 shows expression of GFP by transfected BHK-21cells transfected with the IgG V_(H)-V_(L) fragment.

DAF was then fused to either the CD59sp-V_(H)-V_(L) orIgGsp-V_(H)-V_(L). The CD59sp-V_(H)-V_(L)-DAF is a 537 amino acidpeptide (SEQ ID NO: 34). The IgGsp-V_(H)-V_(L)-DAF is a 534 amino acidpeptide (SEQ ID NO: 35).

BL21 cells were transformed with the pET16b expression vector containingthe scFv-DAF construct as previously described. Expressed protein waspurified using a Hitrap chelating HP column (manufacturer) and refoldedby urea gradient dialysis. Samples taken during the expression,purification, and refolding steps were analyzed by SDS-PAGE. See, FIG.9. Lane 1 is a sample of thepET16b-scFv-DAF/BL21 before induction; lane2 is a sample of thepET16b-scFv-DAF/BL21 after induction; lanes 3 and 4are eluted peaks of the fusion protein from Hitrap chelating HP columns;lanes 5 and 6 are refolded fusion protein after urea gradient dialysis;lane M is molecular weight markers.

Example 5

The scFv-DAF fusion protein was analyzed for specificity binding tohAChRα1-210 peptides by ELISA.

hAChRα1-210 peptides (2 μg/ml) were coated on plates and incubated withserially diluted scFv-DAF or scFv1956. Results were expressed in ODs.Results are presented in FIG. 10. Values represented the mean±SD.*P<0.05.

Example 6

The scFv-DAF fusion protein was analyzed for in vitro complementregulatory function.

Antibody-sensitized sheep erythrocytes were used as target. The degreeof complement-mediated haemolysis was quantified by the release ofhaemoglobin to the supernatant and plotted as molar concentration ofinhibitor present in the assay. Results represent the man value±SD ofexperiments carried out in triplicate. Results are presented in FIG. 11.*P0.05.

Example 7

In this Example, targeting of scFv-35-DAF to the neuromuscular junctionwas analyzed.

The scFv-35-DAF prepared in Example 6 was injected into C57/Black 6 andCD59−/−, DAF−/− mice, which are highly susceptible to complement injury.After 24 hours weight and weakness were assessed. The animals were foundto gain weight and exhibited no loss of strength indicating thescFv-35-DAF did not inhibit neuromuscular junction transmission.Diaphragms were used to visualize localization of the scFv-35-DAF to theneuromuscular junction by the anti-Rat IgG and Bungarotoxin to identifyjunctions. As shown in FIG. 12, the scFv-35-DAF was localized to theneuromuscular junction in both animals and there was no evidence oftissue destruction, even in the complement regulator deficient mouse.These results confirmed that the scFv-35-DAF construct is safe andspecifically targets to the neuromuscular junction.

The scFv-35-DAF was also administered to Lewis rats. The acetylcholinereceptor antibody was administered to the Lewis rats 24 hours later toinduce experimental myasthenia gravis. At 48 hours, the severity ofweakness was maximal in control rats. Of five animals treated withscFv-35-DAF, all survived and showed mild-to-moderate weakness. In therats treated with scFv-35, 3 animals died and 2 had severe weakness.These results indicated that scFv-35-DAF was safe and has a robustprotective effect.

Example 8

In this Example, the affect of scFv-35-DAF on the deposit of C3 on TE671cells was analyzed.

TE671 cells were treated with normal basal medium with scFv-DAF,scFv1956, and DAF (100 nM). Negative controls were treated with PBS inplace of mAb35. Cells were stained with FITC-conjugated anti-C3 antibodyand stained with Eosin staining solution as contrast stain. Images wereobtained at 200× (Panels A-E), 400× (Panels F-J), and 1000×(Panels K-O).Cells were also analyzed by flow cytometry.

Results are presented in FIG. 13. Staining of C3 deposited on cellstreated with scFv-DAF and scFv1956 was patchy and moderate (see, PanelsL and M). The staining of C3 deposits around TE671 cells was diffuse(see, Panels M, N, and O). Scale bar=10 μm.

Results demonstrated that scFv-DAF fusion protein inhibits C3 depositionon the TE671 cell surface.

Example 9

In this Example, the therapeutic effect of scFv-35-DAF in EAMG mice andrats was analyzed.

Specifically, EAMG was induced in DAF−/−, CD59ab−/− mice and Lewis ratsfollowed by treatment with scFv, scFv-DAF, and PBS (as a control). Allmice showed no evidence of disease (see FIG. 15), while rats treatedwith scFv-DAF showed significant protection from EAMG (FIG. 16).Quantitative analysis of complement deposition demonstratedsignificantly less MAC deposition at endplates of scFv-Daf-treatedanimals compared to both vehicle- and scFv-treated rats. In comparingthe scFv and the vehicle, the scFv had a marginally significant increasein complement deposition. Consistent with the better clinical outcome,AChR density was significantly better in the scFv-DAF-treated rats thanscFv and vehicle treated rats (not shown).

In view of the above, it will be seen that the several advantages of thedisclosure are achieved and other advantageous results attained. Asvarious changes could be made in the above processes and compositeswithout departing from the scope of the disclosure, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

When introducing elements of the present disclosure or the variousversions, embodiment(s) or aspects thereof, the articles “a”, “an”,“the” and “said” are intended to mean that there are one or more of theelements. The terms “comprising”, “including” and “having” are intendedto be inclusive and mean that there may be additional elements otherthan the listed elements.

1. A composition comprising a neuromuscular junction targeting peptidecoupled to a therapeutic agent.
 2. The composition of claim 1, whereinthe neuromuscular junction targeting peptide comprises a lamininpeptide, a human immunodeficiency virus nucleocapsid zinc finger domain,a rabies virus glycoprotein peptide, an α-bungarotoxin peptide, an agrinpeptide, a single chain antibody peptide that specifically binds toacetylcholinesterase and a single chain antibody peptide thatspecifically binds to acetylcholine receptor.
 3. The composition ofclaim 1, wherein the neuromuscular junction targeting peptide comprisesan amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ IDNO:
 9. 4. The composition of claim 1, wherein the therapeutic agent isselected from the group consisting of a complement inhibitor, anacetylcholinesterase inhibitor, a trophic agent, and a paralytic agent.5. The composition of claim 4, wherein the complement inhibitor is decayaccelerating factor, rEV, rEV576, membrane cofactor protein, compstatin,a compstatin derivative, POT-4, a Cl inhibitor, C4b-binding protein,factor H, complement receptor Ig, CD59, clusterin, a C3-inhibitor,peptide 2J, human beta-defensin 2, CRIT-H17, Ac-SHLGLAR-H, Ac-RLLLAR-H,C1s-INH-248, S-protein, Crry, circumin, W-54011, NDT9520492, NGD 2000-1,CP-447,697, NDT 9513727, SB290157, SB290157(A), SB290157(B), BCX1470,PMX53, PMX205, C089, and JPE1375.
 6. The composition of claim 1, furthercomprising a linker.
 7. The composition of claim 6, wherein the linkeris selected from the group consisting of a glutamine-serine linker, SEQID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23,SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO:28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQID NO:
 33. 8. A method of delivering a therapeutic agent to theneuromuscular junction, the method comprising: administering acomposition comprising a neuromuscular junction targeting peptidecoupled to a therapeutic agent.
 9. The method of claim 8, wherein theneuromuscular junction targeting peptide comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO:
 9. 10. Themethod of claim 8, wherein the therapeutic agent is selected from thegroup consisting of a complement inhibitor, an acetylcholinesteraseinhibitor, a trophic agent, and a paralytic agent.
 11. The method ofclaim 8, wherein the composition further comprises a linker.
 12. Themethod of claim 11, wherein the linker is selected from the groupconsisting of a glutamine-serine linker, SEQ ID NO: 19, SEQ ID NO: 20,SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO:25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ IDNO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO:
 33. 13. A methodfor treating a neuromuscular junction-related disease or condition in asubject in need thereof, the method comprising: administering to thesubject a therapeutically effective amount of a composition comprising aneuromuscular junction targeting peptide coupled to a therapeutic agent.14. The method of claim 13, wherein the neuromuscular junction-relateddisease or condition is selected from the group consisting of myastheniagravis, experimentally acquired myasthenia gravis, Lambert-Eatonsyndrome, Miller Fischer syndrome, congenital myasthenic syndromes,botulism, and organophosphate poisoning.
 15. The method of claim 13,wherein the neuromuscular junction targeting peptide comprises an aminoacid sequence selected from the group consisting of SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO:
 9. 16.The method of claim 13, wherein the therapeutic agent is selected fromthe group consisting of a complement inhibitor, an acetylcholinesteraseinhibitor, a trophic agent, and a paralytic agent.
 17. The method ofclaim 16, wherein the complement inhibitor is selected from the groupconsisting of DAF, rEV, rEV576, membrane cofactor protein, compstatin, acompstatin derivative, POT-4, a Cl inhibitor, C4b-binding protein,factor H, complement receptor Ig, CD59, clusterin, a C3-inhibitor,peptide 2J, human beta-defensin 2, CRIT-H17, Ac-SHLGLAR-H, Ac-RLLLAR-H,C1s-INH-248, S-protein, Crry, circumin, W-54011, NDT9520492, NGD 2000-1,CP-447,697, NDT 9513727, SB290157, SB290157(A), SB290157(B), BCX1470,PMX53, PMX205, C089, and JPE1375.
 18. The method of claim 13, whereinthe administering step is by infusion, injection, orally, nasally,topically, and subcutaneously.
 19. The method of claim 13, wherein thetherapeutically effective amount has a half maximal inhibitoryconcentration of from about 0.001 μg/ml to about 40 μg/ml.
 20. Themethod of claim 13, wherein the composition has a binding affinity offrom about 0.5 nM to about 50 μM.